An immunotoxin is a chimeric molecule which combines a cell surface binding region, such as from an immunoglobulin domain, and a toxin region typically derived from a naturally occurring protein toxin, such as those found in bacteria or plants. The potency of an immunotoxin greatly depends on its efficiency in transiting from the cell surface to the cytosol, a process that begins with cell internalization (see Pirie C et al., J Biol Chem 286: 4165-72 (2011)).
CD20 is a member of a family of polypeptides known as the membrane-spanning 4A (MS4A) family that includes at least 26 proteins in humans and mice (Ishibashi K et al., Gene 264: 87-93 (2001)). As with all MS4A members, the CD20 sequence predicts three hydrophobic regions forming a transmembrane molecule that spans the membrane four times, a structural characteristic believed central to its function. Also predicted is a single extracellular loop between the proposed third and fourth transmembrane domains and intracellular amino- and carboxy-terminal regions (Tedder T et al., Proc Natl Acad Sci 85: 208-12 (1988)). It is within this extracellular loop of approximately 40 amino acids that the majority of anti-CD20 monoclonal antibodies (mAbs), such as rituximab, are believed to bind with alanine-170 and proline-172 being the most critical residues. A crystal structure of an antibody binding a peptide fragment of CD20 using amino acids 163-187 of CD20 has confirmed amino acids 170 (alanine) through amino acids 173 (serine) as antigen-antibody interaction points for rituximab and CD20 (Du J et al., J Biol Chem 282: 15073-80 (2007)).
CD20 is believed to be present on the cell surface as a homo-multimer, likely a tetramer, and electron microscopy has shown that 90% of complexed CD20 is present in the membrane in lipid rafts and microvilli (Li H et al., J Biol Chem 279: 19893-901 (2004)). Lipid rafts are micro-domains found in the plasma membrane which have high polypeptide, sphingolipid, and cholesterol concentrations (Brown D, London E, Annu Rev Cell Dev Biol 14: 111-36 (1998)). Microvilli, or microvillar channels, are cell extensions from the plasma membrane surface (Reaven E et al., J Lipid Res 30: 1551-60 (1989)). Some antibodies to CD20 are known to bind only when the molecule is present in lipid rafts, such as FMC7 (Polyak M et al., Leukemia 17:1384-89 (2003)) and others, such as rituximab, are known to increase association of CD20 to rafts (Li H et al, supra). It is hypothesized that raft association is important to the proposed function of CD20 as an amplifier of calcium signals that are transduced through the B-cell antigen receptor (BCR), another protein commonly located within lipid rafts and found associated with CD20 multimers (Polyak M et al., J Biol Chem 283: 18545-52 (2008)).
Antibody-based therapies targeting a CD20 antigen are numerous (see Boross P, Leusen J, Am J Cancer Res 2: 676-90 (2012), for review). One of the attractive characteristics of CD20 as a target for therapies based on a mechanism in which the therapeutic remains on the cell surface in order to function is the lack of CD20 cellular internalization after being bound by antibody-based therapeutics (Anderson K et al., Blood 63: 1424-33 (1984); Press 0 et al., Blood 69: 584-91 (1987)). Although this has proven to be both cell-type and antibody-type specific, in general, CD20 appears to internalize at a much lower rate than do other cell surface antigens (Beers S et al., Sem Hematol 47: 107-14 (2010)).
There is a question in the art as to the utility of CD20 antigens as a target for therapies that require the therapeutic to internalize into a target cell after binding in order to be effective because of the general finding that CD20 does not readily internalize (Anderson K et al., Blood 63: 1424-33 (1984); Press 0 et al., Blood 69: 584-91 (1987); Beers S et al., Sem Hematol 47: 107-14 (2010)). Thus, there is an unsolved problem in targeting CD20 antigens with immunoglobulin-type therapeutics that require cell internalization for efficacy—how to force the CD20 bound therapeutic into the target cell's interior after binding. For example, therapies based on the delivery of an immunotoxin that targets a CD20 antigen are predicted to be ineffective based on insufficient CD20 internalization efficiency. Thus, there is a need in the art to develop effective compositions, therapeutics, and therapeutic methods that target cell-surface antigens which do not natively internalize at an efficient rate or upon binding by an immunoglobulin-type domain, like CD20.
In particular, there remains a need in the art to identify and develop CD20-targeted compositions that trigger rapid and efficient cellular internalization of the complex of the composition bound to CD20. For example, cytotoxic CD20-binding proteins comprising toxin-derived regions that induce cellular internalization of native CD20 molecules are desirable for the development of effective cancer and immuno-modulatory therapeutic molecules that target cells of B-cell lineages.